An Overview of Growth,
Aging, Senescence, and Immortality in our HMEC Culture System

Introduction

The goal
of our studies since 1976 has been to understand the normal processes governing
growth, aging, and senescence of HMEC, and how these normal processes are
altered during immortal and malignant transformation. To address these goals, our
labs have generated a large variety of HMEC types, ranging from primary
organoid material and normal HMEC strains, to isogenic series of cultured cells
at various stages of transformation (Chart
1).
Examination of these cultures has elucidated the many significant differences
between normal and abnormal HMEC, and produced a new, molecularly defined model
of the senescence barriers encountered by cultured HMEC (Figure
1, Table 1). These senescence barriers
function to suppress tumorigenesis, thus understanding how they are overcome as
normal cells transform to immortality and malignancy can provide insight into
the mechanism of human malignant progression in vivo, and into possible
therapeutic interventions. Our HMEC culture system has been shown to
accurately model many aspects of early stage breast carcinogenesis in vivo, and
can serve as an experimentally tractable model to examine factors that
influence human cellular aging and carcinogenesis. Importantly, our ability to
achieve long-term growth of normal finite HMEC of multiple lineages makes possible
extensive examination of normal HMEC biology, differentiation, and aging. We
believe that understanding aberrant biological process requires prior knowledge
of the normal state.

Model

We
postulate that cultured finite lifespan HMEC encounter two mechanistically
distinct barriers to indefinite proliferation, stasis (stress-associated
senescence) and replicative senescence (telomere dysfunction due to telomere
attrition). Finite HMEC are also vulnerable to oncogene-induced senescence
(OIS). Errors are needed to bypass or overcome the stasis and replicative
senescence barriers. Once cells become immortal (telomerase expressing) they
are no longer vulnerable to OIS, and a gain-of-function oncogenic error can be
sufficient to confer malignant properties.

Fig.1.
Model of senescence barriers encountered by cultured HMEC

Table.1. Molecular properties of cultured HMEC at senescence barriers

Stasisis a
stress-associated barrier, mediated by the retinoblastoma (RB) pathway, that is
independent of telomere length and extent of replication. The onset of stasis
in cultured HMEC correlates with increased expression of p16,but
not p21 (1-4).
The number of population doublings (PD) achieved prior to stasis depends upon
culture conditions; we have observed a range of ~10-60 PD (3-6).
Molecular correlates that can identify stasis, in addition to p16 expression,
include arrest in G1, low labeling index (LI), non-critically short telomeres
and normal karyotypes (2-4).
These parameters are consistent with an RB-mediated arrest and the absence of a
significant DNA damage response (DDR). Cells at stasis express
senescence-associated β-galactosidase (SA-β-Gal) activity and a senescent
morphology. Stasis can be bypassed or overcome in cultured HMEC by multiple
types of single alterations (genetic and/or epigenetic) in pathways governing RB,
and does not require loss of p53 function (1,6-9).
Overcoming stasis may correlate with hyperplasia/atypical hyperplasia in vivo,
which commonly display clonal growth and errors in the RB pathway (e.g., loss
of p16 expression, mutated RB, overexpressed cyclin D1) (1,10-13).
Gross genomic aberrations are not common at this stage in vivo (14),
and are not associated with overcoming stasis in vitro (2,4).

We
postulate that stasis can also be enforced by p53-dependent p21 in response to
DNA damaging stresses such as oxidative damage or radiation. Although neither cultured
HMEC or their isogenic mammary fibroblasts express p21 at stasis, other cell
types may be more vulnerable to DNA damage inducing stresses in culture,
express p21, and show greater evidence of a DDR at stasis, including telomeric
foci. HMEC in vivo may also experience p53-inducing stresses. The
p53-dependent type of stasis arrest does not require critically short
telomeres or genomic instability, and inactivation of p53 (or p21) function may
facilitate overcoming this arrest (15,16).
Reactivation of telomerase is neither necessary nor sufficient to overcome
stasis.

Replicative senescence occurs
in post-stasis HMEC (cells that have bypassed or overcome stasis) due to
ongoing proliferation producing progressively shortened telomeres, in the
absence of sufficient telomerase activity. When telomeres become critically
short (mean TRF ≤ 5 kb), genomic instability and a DDR is elicited. Where
wild-type p53 is present, most cells show a viable arrest; this barrier has
been termed agonescence(2,3,17).
Karyotypic analysis of HMEC at agonescence has shown that virtually all
metaphases exhibit gross chromosomal abnormalities, predominantly telomere
associations (2).
This result is not consistent with a hypothesis that a p53-dependent senescence
arrest due to telomere attrition occurs as soon as one uncapped telomere is
present (18,19).
When p53 is non-functional a viable arrest is not possible, and crisis-associated
massive cell death occurs (3).
Agonescence can be distinguished from stasis in HMEC by the presence of
critically short telomeres and genomic instability, higher LI (~15%), arrest at
all phases of the cell cycle, and presence of a DDR (Table 1). HMEC at
agonescence as well as at stasis display a senescent morphology and SA-b-Gal,
so these properties do not readily distinguishable between these two molecularly
distinct senescence barriers. Crisis can be distinguished from agonescence in
HMEC by a higher LI (~40%) and the absence of a viable arrest. Since most
human epithelial and fibroblast cells induced to transform to immortality in
culture had inactivation of p53 function to facilitate overcoming stasis (e.g.,
using viral oncogenes or inhibitors of p53 function), only crisis was observed
in such cultures at replicative senescence.

The
barrier due to telomere attrition can be overcome by the expression of
sufficient telomerase to maintain stable telomere lengths. Overcoming replicative
senescence may correlate with DCIS in vivo, which commonly display short
telomeres, genomic instability, and telomerase reactivation (14,20).
That most DCIS contain critically shortened telomeres indicates that the
precursor cells did not express sufficient telomerase for telomere maintenance.

Cultured
finite lifespan HMEC are vulnerable to oncogene-induced senescence (OIS) (21,22).
HMEC that have attained immortality via reactivation of endogenous telomerase
are no longer vulnerable to OIS, and show gain of malignancy-associated
properties when exposed to oncogenes such as Raf-1, Ras or ErbB2 (21,23,24).
HMEC immortalized by exogenous hTERT transduction may appear initially sensitive
to OIS, but can maintain growth (25).
The molecular correlates of OIS in HMEC differ from those seen in cells at
stasis or telomere dysfunction (Table 1). OIS in HMEC does not require p16
or p53 function, and is independent of telomere length; its molecular
properties are consistent with a DDR (21,22).

Finite Lifespan HMEC: Pre-stasis and Post-stasis

HMEC
derived from reduction mammoplasties, milk, benign tumors, and non-tumor
mastectomy tissues have been grown in either serum-containing (MM, M85, M87A)
or serum-free (MCDB 170) media (Chart
1)(4-6,26,27). Depending upon the media and culture conditions,
active proliferation has ceased after ~10-60 PD (Figure 2). In media that
support fewer PD, levels of p16 expression increase earlier; virtually all
cells express p16 at stasis in all media used (1,4). The molecular profile of the
HMEC at stasis is similar regardless of their PD potential or growth media
(Table 1), with one noticeable difference. HMEC grown in serum-containing
media have a typical senescent morphology of large flat vacuolated cells,
whereas HMEC that had been grown in serum-free MCDB170 exhibit a more elongated
morphology showing abundant stress fibers (4,6,27).
We believe this difference is due to the serum-free medium being more stressful
for cultured HMEC, consistent with the early rise of p16 and the low PD
potential of HMEC initiated in MCDB170 (commercial MEGM [Lonza] and M171 [Life
Technologies] are based on our original MCDB170) (1,6).
This difference in morphology may have led other investigators to consider this
stasis arrest distinct, and refer to it as “M0” (28,29).

Pre-stasis in M87A: Our most recently developed medium,
M87A, supports long-term growth of the normal pre-stasis HMEC (Figures 2,3) (4).
The pre-stasis HMEC we now distribute were grown in this medium. Populations
contain a mixture of cells with markers of myoepithelial, luminal, and
progenitor lineages; later passage cultures show fewer luminal cells (30,31).
While not yet carefully examined, cells with luminal markers may also cease proliferation
as a consequence of terminal differentiation rather than p16(+) stasis. Senescent
cells remain genomically stable. We have examined gene transcript profiles,
global promoter methylation, and DDRs as a function of passage from several
individual’s HMEC (4,9). As expected, gene expression
changes significantly with passage, while no obvious differences were seen for
promoter methylation. Some interindividual differences could be detected in
gene expression and extent of DDRs. Previous studies have shown
interindividual differences in carcinogen metabolism (32),
leading us to recommend that at least 2 individuals be examined to determine
normal HMEC properties. We have also observed differences in lineage markers and
differentiation correlated with the age of the specimen donor (14,30). With increasing age, HMEC from
4th passage pre-stasis strains and from uncultured dissociated
organoids showed a decline of myoepithelial
cells, and an
increase of luminal
cells that exhibited
molecular features usually ascribed to myoepithelial
cells (increased expression of integrin a6
and keratin (K)14).
The proportion of c-Kit expressing cells (putative progenitors) also increased with age, and exhibited impaired microenvironment-directed
differentiation and lineage specificity, consistent with alterations in Hippo
pathway activation. These data suggest that the observed age-associated
increase in luminal breast
cancer could be connected to changes that occur normally with aging in the
human breast. Myoepithelial
cells are thought to be tumor-suppressive and progenitors are putative
etiological roots of some breast cancers. Thus during the aging process, the
potential target cell population may increase while there is a simultaneous
decrease in the cells thought to suppress tumorigenic activity.

Figure 2. Population doubling potential of pre-stasis HMEC in
different media. Primary cultures from reduction mammoplasty specimen 184 were
initiated from organoids in different media. The number of PD in primary
culture cannot be accurately determined; growth is shown starting from passage
2. All proliferation stopped in HMEC grown in serum containing media (MM and
M87A with oxytocin (X)) after 10-50 PD beyond passage 2. The extensive
proliferative potential in M87A+X supports generation of large batches of early
passage pre-stasis HMEC from individual donors. HMEC initiated in serum-free
MCDB 170 (commercial MEGM) show rapid induction of p16 and cessation of
growth. When cultures are allowed to sit without subculture for 2-3 weeks,
post-selection post-stasis HMEC emerge and maintain growth to replicative
senescence. If cultures are repeatedly subcultured, fewer to no post-selection
cells may emerge.

Figure 3. Characterization of pre-stasis HMEC grown in M85 with oxytocin. A. Expression of markers associated with proliferation (LI) and senescence (p16, SA-β-Gal) in pre-stasis 184D HMEC with increasing passage; stasis was at passage 15. Note the reciprocal relationship between the small cells with a positive LI, and the larger, often vacuolated cells (senescent morphology) that are positive for p16 and SA-β-Gal, and negative for LI. B. Immunohistochemistry expression of luminal lineage marker K19 in pre-stasis 184D HMEC. C. Immunofluorescence expression of luminal lineage markers EpCam and Muc1 in pre-stasis 48RT HMEC. Size marker = 200 microns.

Pre-stasis and post-stasis in MM: We have cultured pre-stasis
HMEC from over 150 individuals in serum-containing media and have not observed
even a single instance of a cell spontaneously overcoming the stasis barrier. However,
early experiments that exposed primary cultures of specimen 184 HMEC grown in
MM to the chemical carcinogen benzo(a)pyrene (BaP) resulted in the emergence of
HMEC colonies that maintained growth after the bulk of the cultures ceased
proliferation at stasis (7,33).
These BaP post-stasis populations (originally
called Extended Life), ceased growth
after an additional 10-40 PD, with the exceptional of very rare cells that
became immortal cell lines (see below). All three BaP post-stasis cultures that
have been examined showed loss of p16 expression, associated either with
mutation or promoter silencing (1,13).
Whole exome analysis (34)
indicates that these cultures contain ~100-200 BaP-induced point mutations, the
majorityconsistent
with the known mutation spectrum of BaP. We
have very limited quantities of BaP cultures available for distribution on a
collaborative basis.

Pre-stasis and post-stasis in MCDB170: When the HMEC are cultured in
the highly stressful serum-free MCDB170 medium, a small number of cells are
able to overcome stasis in the absence of additional oncogenic exposures (6)
(Figure 2). These post-stasis cells show methylation of the p16 promoter and
absence of p16 expression, as well as nearly 200 other mostly cancer-associated
changes in promoter methylation (in contrast to the BaP post-stasis cultures,
which display only ~10 changes) (1,9). We originally called the
emergence of these post-stasis cells “selection” and this class of post-stasis HMEC “post-selection”.
We now recognize that selection (what other labs latter termed “M0”) is a
stasis arrest. Although the pre-stasis populations may be heterogeneous with
respect to a cell’s ease in silencing p16 to become post-selection (35),
we believe the post-selection cells are induced by growth in the stressful (oncogenic)
serum-free MCDB170 medium, i.e., post-stasis cells are not present in the
starting normal pre-stasis cultures. This is based on the total absence in over
30 years of our work of any post-stasis cell emerging from normal pre-stasis
HMEC grown in any of our serum-containing media, as well as the absence or
reduction of post-selection HMEC emerging from pre-stasis HMEC grown in MCDB170
when there are small changes in media composition (e.g., absence of a cAMP
stimulator) or methodology (e.g., sub-culturing cells approaching stasis/selection
rather than waiting 2-3 weeks at stasis without subculture for the
post-selection cells to emerge; we presume the induction of the p16(-) cells is
occurring during this time when no cell divisions are observed). It is likely
that post-stasis cells exist in some breast tissues; p16(-) HMEC have been seen
in apparently normal breast tissues in vivo (35).
These rare cells have been called vHMEC; the nature of the error(s) leading to
the silencing of p16 in vHMEC in vivo is not known; the term vHMEC has also
been used by others to refer to p16(-) post-stasis cells in culture that are
specifically post-selection. Of note, it has been suggested that the aberrant
post-selection post-stasis HMEC (which are sold commercially as normal primary
HMEC, e.g., Lonza CC-2551 and Life Technologies A10565) may be on a pathway to
metaplastic cancer (36).

Post-selection
post-stasis p16(-) HMEC grow actively for an additional ~30-70 PD, depending on
the individual. They express wild-type p53 that is present in a stable form (3,37,38).
As they near agonescence, they exhibit a senescent morphology, SA-β-Gal, a DDR, and genomic
instability (2,3). If p53 function is inactivated
(e.g., using the genetic suppressor element GSE22 (3W9))
cells continue to proliferate for an additional ~2-4 passages, with increasing
evidence of cell death and debris (i.e., crisis) (Figure 4)(3).
The telomere dysfunction barrier is very stringent in post-selection post-stasis
HMEC. We have never seen any unperturbed cell at agonescence spontaneously
immortalize. We have also never seen any immortalization at crisis in
post-selection HMEC, but rare immortalization at crisis using DN-p53 constructs
has been reported by others (40,41).
This stringency is due in part to the molecular nature of this barrier; cells
that fail to maintain a G1 or G2 arrest with critically short telomeres will
eventually die or become non-proliferative as a consequence of the genomic
instability and mitotic catastrophes. Overcoming replicative senescence requires
induction of sufficient telomerase; in post-selection post-stasis cells
(see more below), more than one error is needed to reactivate telomerase. The
probability of this occurring in one p53(+) cell during telomere dysfunction is
exceedingly low, accounting for the lack of immortalization in the
post-selection cultures at agonescence.

We have
large supplies of post-selection HMEC available for distribution from women of
various ages. It’s important to recognize that these cells are not normal, and
acquire genomic instability as they are propagated in culture. In the past,
due to the inability to attain long-term culture of normal pre-stasis HMEC, we
provided post-selection HMEC for studies on finite HMEC. Since it is now possible
to grow large quantities of normal pre-stasis HMEC, we recommend that studies
aiming to understand normal HMEC behavior use normal HMEC and not the aberrant
post-selection HMEC. For some experimental purposes, post-selection or other
post-stasis HMEC may be preferable, e.g., examining the requirements for and
mechanisms of overcoming the telomere dysfunction barrier, or assaying cells at
different stages in progression.

Figure 4. Growth and morphology of post-stasis post-selection 184 with and without functional p53. 184B HMEC were transduced with GSE22-containing or control (Babe) vectors at passage 5. (A) growth curves of 184B-Babe and 184B-GSE22. Note the additional PD in the cultures lacking functional p53. We believe growth rates are similar ± p53, but the absence of p53-mediated growth inhibition allows more cells to continue to proliferate to crisis, leading to apparent faster growth of the population as cells near telomere dysfunction. (B) 184B-Babe at agonescence, 2 months after plating at passage 15, contains mostly large, flat cells with some vacuolization; the cell population can retain this morphology and viability for over a year. (C) 184-GSE22, two weeks after plating at passage 15, shows areas of small proliferating cells and many very large flat cells (arrows). (D) 184B-GSE22, four months after plating at passage 15, shows mostly large multi-nucleated, vacuolated cells and abundant cell debris. All photographs are at the same magnification. (3)

Post-stasis in M85 or M87A: More recently, we have generated additional types
of post-stasis HMEC using shRNA to p16 (p16sh) (42) and transduction of a cyclin D1/CDK2
construct (D1) (43). As
expected, in early passage HMEC grown in M85 or M87A, direct inhibition of p16
using p16sh, or transduction of D1, led to widespread bypass of stasis. Post-stasis
p16sh HMEC show few differences in their pattern of promoter methylation or
gene transcripts compared to their precursor pre-stasis cultures ((1,9)unpublished). They grow for an
additional ~20-30 PD until agonescence (Figure 5). Unlike post-selection post-stasis,
and similar to BaP post-stasis cells, p16sh post-stasis HMEC have generated
rare clonal immortal lines during the period of genomic instability at
agonescence. This difference (see more below) is presumably due to the ability
of just one error to immortalize these cultures (42).
We can provide limited amounts of the p16sh post-stasis HMEC for specific
requests.

I want
to add a few comments about nomenclature since this issue has frequently come
up in discussions. It’s my general experience in science that functionally
distinct molecules or molecular processes are given distinct names. Confusion
could result if there were not distinct names for different, though closely
related family members (e.g., growth factors and their receptors) or related
mechanisms (e.g., apoptosis, anoikis, mitotic catastrophe). One of our overall
goals is to try to model the many different in vivo pathways a normal cell can
take to become malignant. Such information may assist individualized clinical
interventions. Our data thus far indicate that molecular properties differ
among different pathways, starting with early stage carcinogenesis. Pre-stasis
HMEC differ from post-stasis HMEC, although both are finite, and as discussed
above, there are significant differences among the various post-stasis types. Since
these different post-stasis cultures are functionally different, we have given
them distinct names. Similarly, since agonescence is molecularly and
morphologically distinct from crisis, although both result from telomere
attrition, we believe it important that there be distinct names. Confusion may
also arise if similar mechanisms are given distinct names, e.g., we view what
we are defining as stasis as having also been referred to as M0, M1, MINT,
M1.5, premature senescence, replicative senescence, and culture shock. Telomere
dysfunction due to telomere attrition (replicative senescence displaying as agonescence
or crisis) has been called replicative senescence, crisis, M2, and M1. Indeed,
it was this situation that prompted our initial efforts to generate molecularly
defined nomenclature for the senescence barriers (2).
I encourage everyone to employ the molecularly defined nomenclature we have
presented here for our HMEC culture system.

Immortally Transformed Cell Lines

The
replicative senescence barrier can be overcome or bypassed by the expression of
sufficient telomerase to maintain stable telomere lengths. However, unlike bypassing
an arrest based upon keeping an active RB (e.g., abrogating the p16 expression
that enforces stasis), the widespread chromosomal derangements caused by telomere
dysfunction are not reversible. Consequently, in cultured HMEC, overcoming
replicative senescence differs from bypassing/overcoming stasis in that the escaped
immortal cells will contain the genomic abnormalities accumulated to
that point, and may retain
some degree of genomic instability (14).
We hypothesized that the inherent genomic instability consequent to telomere
attrition can give rise to the errors permissive for telomerase reactivation, as
well as many of the “passenger”
errors seen in breast carcinomas,and that the
generation of breakage-fusion-bridge (BFB) cycles prior to
immortalization may underlie some of cancer-associated genomic instability (3,44).
Thus many errors
that can contribute to the ultimate cancer cell phenotype, including level of
aggressiveness, may arise prior to immortalization and malignancy. This
hypothesis is consistent with publications indicating that many properties of
invasive tumors are already present in their pre-invasive DCIS lesions, such as
tumor markers, gene expression profiles, gene methylation, PIK3CA mutations,
and genomic errors (45-49).

Our
recent studies on immortalization support these hypotheses, as well as our
model of the HMEC senescence barriers (34,42).
Reactivation of sufficient telomerase in cultured finite lifespan HMEC has been
difficult to achieve using pathological relevant agents (i.e., not ectopic hTERT
or viral oncogenes). Reported immortal lines have been rare clonal events (3,7,8,40-42,50-52).
This likely reflects the fact that large long-lived animals such as humans have
evolved mechanisms for stringent repression of telomerase in normal adult
non-stem cells, presumably for tumor suppression. In contrast, cells from
small short-lived animals such as mice do not show such stringent telomerase
repression, and, lacking the replicative senescence barrier, readily
immortalize once they overcome stasis (53-55).
We believe that immortalization with telomerase reactivation is a rate-limiting
step in human epithelial carcinogenesis, and thus great caution should be
exercised in extrapolating mechanisms of rodent malignant progression to
humans. One of the goals of our long-term program in developing an HMEC model
system of transformation has been to make available experimentally tractable human
cells for examination of this crucial step in human malignant progression,
since it cannot be accurately modeled in mice (42).
As described further below, our initial work generated rare clonally
immortalized lines containing multiple genomic errors, making it difficult to examine
the immortalization process. Our recent work has shown that efficient
non-clonal immortalization is possible when the two main senescence barriers
(stasis and replicative senescence) are directly targeted; resulting
immortalized lines display normal karyotypes. These new methods should allow
experimental examination of the immortalization process in HMEC.

We have
generated a variety of immortally transformed lines using various oncogenic
agents (see: Chart
1and Cell Types Generated) (3,7-9,21,33,42,50,56-59).
Most of these lines were derived from post-stasis cultures, although in a few
instances (involving hTERT or c-Myc transduction) lines emerged from
perturbations of pre-stasis populations. Our first immortal lines were
obtained from the BaP post-stasis cultures, 184Aa, 184Bd, and 184Be (7,8,33,56).
Extremely rare immortal lines have appeared at agonescence (184A1, 184AA4, 184AA8,
184B5, 184BE1, 184BE2). The BaP post-stasis cultures harbor small mutations from
their BaP exposure, as well as loss of p16 expression (34).
Large genomic errors such as copy-number variations (CNV) were seen in examined
immortal lines but not the precursor BaP post-stasis cultures (34,60). Presumably, the genomic
instability at agonescence produced the CNV and the rare errors that allowed
telomerase reactivation and immortalization. More frequent but still rare
clonal lines appeared at agonescence following transduction of the breast
cancer–associated oncogene ZNF217 into the 184Aa population (184AaZN1-3 (50)).
More frequent immortal clonal outgrowths at crisis were seen when p53 was
inactivated in 184Aa using GSE22 (184AaGS1-2). All examined (karyology and/or
aCGH) clonal lines exhibited numerous large genomic errors. Uniform
immortalization was obtained following transduction of c-Myc into three
different BaP post-stasis cultures (184AaMY1-5, 184BeMY, 184CeMY (42), indicating that one error is sufficient
to immortalize these BaP post-stasis cultures.

No
post-selection post-stasis HMEC has been observed to spontaneously immortalize,
presumably due to the need for two errors for immortalization. Rare immortal
lines have appeared at agonescence following overexpression of the breast cancer associated oncogene ZNF217 (184ZN4-7)(42,50).
Transduction of c-Myc alone produced 1 clonal line in
10 independent experiments (184SMY1). We hypothesize that rare errors
generated by the genomic instability at agonescence may complement ZNF217 or c-Myc
to allow telomerase reactivation. Overexpression of
both c-Myc and ZNF217 in post-selection HMEC was able to produce clonal
immortal lines in repeat experiments (184ZNMY1-4,
unpublished); some of these clonal lines
immortalized early, prior to agonescence.

More recently, we have tested our hypothesis about the two main
senescence barriers by targeting these barriers in early passage pre-stasis
cells grown in M85 or M87A (42).
If we were correct in postulating that the genomic errors seen in clonal
immortalized cells (in vitro and in vivo cancer) were required to
bypass/overcome these barriers, but genomic instability was not needed per se, then directly targeting
these barriers should produce non-clonal immortalized lines lacking gross genomic
errors. That is indeed what we have observed, using primary HMEC from 4
different specimen donors ranging in age from 19 to 91 (Figure 5). The stasis
barrier was targeted using p16sh or D1/CDK2, and the replicative senescence
barrier was targeted by transduction of c-Myc. Efficient immortalization has
been seen with all specimens in all experiments (currently 8 independent).
Karyotypes obtained from 6 different immortalized lines (184Dp16sMY,
240Lp16sMY, 240LD1, 122Lp16sMY, 122LD1, 805Pp16sMY) were all normal diploid at
early passage. From visual observation, there could be <100% transition to
post-stasis using either p16sh or D1 alone, while the transition to immortality
from the post-stasis cultures appeared close to 100%. c-Myc transduction of the
BaP post-stasis cultures also appeared to give close to 100% immortalization
efficiency. This transduction was done in 184Aa and 184Be after the start of
telomere dysfunction, so the immortalized lines do not exhibit a normal
karyotype, while 184Ce, transduced earlier, generated 184CeMY with a normal
karyotype (tho with many BaP-induced point mutations). The ability of the
p16sh, D1, and BaP post-stasis cells to be immortalized by one error (i.e.,
c-Myc) likely accounts for the rare “spontaneous” immortalization seen in these
cultures at agonescence (184Fp16s, 184Dp16s, 240Lp16s, 805Pp16s and the
BaP-exposed lines). Amplification at the c-Myc region was observed in some of
these clonal lines. We do not know why the post-selection post-stasis cells
differ in this respect. We hypothesize that their prior experience of high
stress may play a role. Transduction of c-Myc alone into pre-stasis HMEC in low
stress-media gave rare clonal escape from stasis; these “post-stasis” cells maintained
growth to became clonal immortal lines (184FMY2, 184DMY3, 240LMY, 122LMY). All clonal
lines examined showed multiple CNV by aCGH. These new lines are still being
characterized and are available upon specific request.

Our studies
have led us to propose the existence of a previously unreported step in HMEC
immortalization, termed conversion, based on observing the need for multiple
steps to attain a fully immortal potential (i.e., synthesizing sufficient
telomerase to maintain stable telomere lengths) (3,8,21,42,56,59)(8,21,50,56-59,61,62). Even after HMEC have acquired
the errors allowing them to bypass/overcome both stasis and replicative
senescence, and express hTERT, the resultant cells with indefinite
proliferative potential still progress through further changes. Conversion is
most prominent in cells that immortalize while retaining functional p53.
Consequently, this process has not been widely studied, as most in vitro
immortalized human epithelial and fibroblast cells had p53 inactivated prior to
immortalization.

Conversion has been most extensively studied in the immortal 184A1
line, which first appeared ~passage 8 in the 184Aa BaP post-stasis population,
and had a mean TRF value of ~5 kb when first examined at passage 11 (Figure 6).
We noted that cells that overcame agonescence gained the potential to express
telomerase, but initially displayed little telomerase activity, and had ongoing
telomere erosion with proliferation. When telomeres got extremely short (<3
kb), the conversion process ensued. Expression of the CKI p57Kip2
initially abruptly increased and then slowly declined, associated with initial
slow-heterogeneous growth and then gradual re-attaining of uniform good
growth. Telomerase activity gradually increased, and the faint very short
telomeres seen during conversion gradually became stabilized with a mean TRF of
~3-7 kb. As telomerase activity increased, the immortal lines gradually
developed the ability to maintain growth in the presence of TGFb; this change is a direct consequence of the hTERT expression, as
transduction of hTERT into post-selection HMEC confers the ability to maintain
growth in TGFb in addition to
producing uniform immortalization (58). A significant change that is associated with conversion and
telomerase expression but not initial immortal potential is the loss of
vulnerability to OIS (21), likely related to a role of telomerase in providing resistance
to OIS (63).

Figure 6. Conversion of newly immortal p53(+) HMEC lines is associated with changes in many key properties. (A.B.) The p53(+) 184A1 line undergoing conversion exhibits changes in growth capacity (CFE) and expression of p57, expression of telomerase activity and mean TRF length, gains the ability to maintain growth in the presence of TGFb, and becomes resistant to OIS (8,21,50,56,61). When pre-conversion 184A1 is transduced with GSE22, there is a rapid increase in telomerase activity associated with stabilization of TRF length (8).

When we obtained immortal HMEC lines that lacked functional p53
(184AA2, 184AA3) we noted that they showed some initial telomerase activity, no
p57 expression, and quickly attained good uniform growth ± TGFb. Their mean TRF length stabilized at ~4-5 kb and never declined
to the very low levels seen in the p53(+) lines. The role of p53 in repressing
telomerase activity in newly immortal lines was then demonstrated by
inactivating p53 (using GSE22) in pre-conversion 184A1 (Figure 6) (8). Endogenous telomerase activity was quickly expressed and mean
TRF lengths stabilized; existing p57 expression was rapidly reduced. GSE22 transduction
into the finite lifespan precursors of the immortal lines did not induce
significant telomerase activity indicating that abrogation of p53 function
alone is not sufficient for telomerase reactivation in post-stasis HMEC. These results
suggest that the newly immortal p53(+) lines have the potential to express
telomerase, but expression is low due to a p53-mediated repression (unpublished
data have also indicated that newly immortal p53(+) lines express low
telomerase activity which can be inhibited). We believe that fully immortal
p53(-) lines are expressing an accelerated but molecularly similar conversion
process as occurs in the p53(+) lines. The resulting cultures express similar
properties, e.g., short stable telomeres, resistance to OIS, and hundreds of
promoter methylation changes (9), but the p53(-) lines never encountered the extremely short
telomeres and p57 expression shown by the p53(+) lines. Newly immortal 184AA2
and 184AA3 did both initially briefly show slower growth and lower TRAP
activity than at later passages.

While we
have gained much information about the molecular properties associated with
conversion, much about this process remains unknown. Our current
speculation is that conversion may reflect a need to alter chromosome
conformation at the telomeres when cells transition from a finite state (no
stable telomere length maintenance) to one where sufficient telomerase
maintains the short stable telomeres. As
well studied in yeast, immortal cells can have “counting” mechanisms to
maintain telomeres within a limited size range (64).
Since most human carcinoma cells, as well as our immortal HMEC lines, maintain
telomeres within a short range (mean TRF ~3-7 kb) (56,65,66),
some type of “counting” mechanism likely is involved. Short stable telomeres
are not seen in normal telomerase expressing human cells such as stem cells and
lymphocytes (67),
suggesting that active processes may be required for conversion to the distinct
telomeric state seen in the immortalized and cancer-derived cells. Functional p53 may
present a partial barrier to the conversion process until very short telomeres
provoke a structural change at the telomeric ends. Since the majority of
breast cancers express wild-type p53, it is possible that the slower p53(+)
version of the conversion process may be relevant to early-stage breast
carcinogenesis in vivo. We have speculated that the low levels of telomerase
expression coupled with extremely short telomeres could make newly immortal
p53(+) breast cancers particularly vulnerable to therapeutic interventions
targeting telomere dynamics.

We, and others, have also immortalized finite HMEC by ectopic
overexpression of the hTERT gene (58,68,69). Initial lines came
from transducing hTERT into post-stasis post-selection HMEC grown in serum-free
medium (184BTERT, 48RTERT, 161TERT); consequently, these immortalized lines
contain the many cancer-associated and other aberrancies found in this p16(-) metaplastic
post-stasis type. We and others were unable to immortalize pre-stasis HMEC
grown in serum-free media with transduced hTERT, and only rare clonal immortalization
occurred when hTERT was transduced into pre-stasis HMEC grown in MM (184FTERT) (58). Hypothesizing that high stress exposure might prevent hTERT immortalization
of pre-stasis HMEC, we transduced hTERT into passage 3 pre-stasis 184D grown in
low-stress M87A, once that medium was available. Nearly uniform immortalization
was observed, generating the non-clonal line 184DTERT1 (42). Caution: TERT reactivation during in vivo
malignant progression is not caused by ectopic introduction. The errors and mechanisms
responsible for TERT reactivation are key to understanding human tumorigenesis
(and its potential prevention), and they cannot be investigated or
recapitulated in lines created by TERT transduction. Further, we have seen
that ectopic TERT overexpression creates lines with properties not found in
normal or abnormal HMEC in vivo. TRAP activity is quite high [unpublished],
compared to more modest levels in most in vitro immortalized and cancer-derived
lines. Where examined, mean TRF length is longer than the typical 2-8 kb range
of immortalized and cancer-derived lines (56,58,66), suggesting that these TERT-immortalized lines did not undergo
conversion and therefore have telomere dynamics distinct from most carcinoma
cells in vivo. Telomerase expression can enable cells to become resistant to
OIS and maintain growth in the presence of TGFβ (21,25,58,63); other signaling
pathways are also affected by transduced TERT (70). 184DTERT did not express and was not inducible for p16
[unpublished]. Based on such data, I do not think that TERT-immortalized HMEC
represent accurate models for normal, aberrant, or cancer (including cancer stem)
HMEC in vivo; rather their expression of in vitro induced artifacts can potentially
obscure understanding of in vivo biology. Since these immortalized lines are
aberrant compared to normal HMEC (which are finite, with no or low TRAP
activity, and with intact stasis, OIS, and replicative senescence tumor
suppressor barriers), they cannot be accurate normal controls.

In
general, we have seen that different methods of producing immortal HMEC, e.g.,
culture medium, oncogenic agent, agent used for direct bypass of stasis, can
yield cell lines with significantly different phenotypes. The age of the specimen
donor may also influence phenotype. Immortalized lines generated from younger
women tend to express a phenotype most similar to the basal subtype of human
breast cancers, while more luminal phenotypes can be seen in lines from older women (43).
The use of the cyclin D1/CDK2 construct to bypass stasis promoted a more
luminal phenotype, even in younger specimens (43). Immortalization of the post-selection
post-stasis types may favor a metaplastic phenotype (36).
Possibly, the prior prevalence of immortalized lines with only a basal or
metaplastic phenotype was a consequence of using cells from mostly younger
women combined with stressful culture medium that did not support growth of
normal HMEC with luminal or progenitor properties. Additionally, in vivo human
malignant progression is influenced by interactions with the microenvironment,
which our current culture conditions do not recapitulate.

Once the HMEC are immortally transformed and no
longer vulnerable to OIS, the introduction of one or two oncogenes can further
transform these cells towards malignancy (anchorage-independent growth, growth
factor independence, and/or tumorigenicity in nude mice) (21,23,24).
Finite lifespan, OIS-sensitive HMEC cannot be rendered malignant by the same
oncogenes. Thus many reports in the literature show that one error can confer
malignancy-associated properties to abnormal, immortally transformed lines such
as MCF10A and 184B5 – because these aberrant lines have abrogated all the
major tumor suppressor mechanisms! Comparisons of non-malignant immortal
lines with oncogene-exposed derivatives that had gained anchorage-independent
growth did not show major differences in gene transcript profiling or global
promoter methylation, in contrast to the major differences seen between all
finite and all immortalized cultures (9,71). These data are consistent with
the acquisition of immortality, rather than the acquisition of malignancy, as the
step in human carcinogenesis most associated with molecular alterations.

Altogether,
these studies validate our model of the senescence barriers encountered by
cultured HMEC, indicate that genomic instability per se is not needed for
immortalization, and support the hypothesis that carcinoma-associated genomic
instability (along with many “passenger” errors) may have its origin in the
inherent instability induced by telomere dysfunction at replicative senescence.
The reproducible ability to generate non-malignant (OIS resistant) immortalized
lines lacking gross genomic errors can facilitate further investigation of the
molecular underpinnings of the immortalization process, and provides substrates
to examine the effects of additional oncogenes and in-vivo identified genomic
alterations on malignant progression.

Note: Immortal HMEC have been
actively transformed to immortality. Normal human somatic cells are
finite, and vulnerable to multiple tumor suppressor barriers (stasis,
replicative senescence, OIS). The stringent replicative senescence barrier to
immortality has evolved in large-long-lived animals like humans to suppress
tumorigenesis. Immortality(expression of sufficient
telomerase to maintain stable telomere lengths)is the most common alteration
from normal associated with human solid cancers. We believe that
attaining immortality is likely the most rate-limiting step in human
carcinogenesis – immortally transformed lines such as 184A1 and MCF10A have
acquired the errors that allowed them to overcome all tumor suppressor
barriers, so that the overexpression of one oncogene can confer
malignancy. Our immortal lines cluster with tumor derived lines and not finite
HMEC in properties such as gene expression, promoter methylation, and
resistance to OIS and TGFb growth inhibition. Immortal
lines may be non-malignant, but they are NOT normal, “normal”, or untransformed.
Please do not refer to immortal HMEC (or any immortalized human cells) as
normal or untransformed. I sometimes despair about how we will be able to
understand and develop therapeutics for early stage human epithelial
carcinogenesis when immortal cell lines such as 184A1 and MCF10A, or
TERT-immortalized post-selection post-stasis HMEC, are routinely referred to in
the literature as normal or untransformed, employed as “normal” controls, or
used as a starting point to study “early stage” carcinogenesis. I view this as
similar to calling telomerase(+) DCIS “normal”, and using it as a normal
control for cancer or the starting point for studying early stage
carcinogenesis. There is already much in the literature to indicate that many,
if not most of the significant alterations seen in breast and other carcinomas
are already present in the pre-malignant stage.